Adenylyl cyclase is a family of enzymes that catalyze the formation of cAMP from ATP. Membrane-bound forms of the mammalian enzyme are stimulated or inhibited by hormones via G-protein-linked cell-surface receptors and are inhibited via a cytoplasmic domain ("P"-site) by specific analogs of adenosine. The proposed investigations will use biochemical and physiological approaches to focus on the regulation of adenylyl cyclase via the "P"-site. Experiments will identify the structural requirements for an the locus of "P"-site-specific ligands synthesized by this laboratory will be determined. b) Radioactive affinity probes, 2'5'dd3'FSB[3H]Ado and 2'5'dd3'A[125I]PPA, will be used to tag the "P"-site domain of adenylyl cyclase. c) Labeled peptides will be isolated and sequenced and sequences will be compared with known structures of adenylyl cyclases and other membrane proteins, e.g. transporters or channels, that may have "P"-site consensus sequences. d) Antibodies to these and other selected peptides will be prepared. e) These antibodies will be used to evaluate cloned and expressed enzymes. And f) additional radioactive and fluorescent probes for the "p"-site and catalytic domains will be developed and used to measure interactions and distances between the two binding domains. One of the most important questions with regard to "P"-site-mediated inhibition of adenylyl cyclase is its physiological relevance. Inhibition of adenylyl cyclase that is coordinated with gene expression, RNA processing, or other aspects of nucleic acid metabolism, are attractive possibilities. To ascertain whether and to what extent inhibition of adenylyl cyclase via the "p"-site is regulated physiologically, investigations will: a) determine whether there are naturally occurring "P"-site agonists and/or antagonists more potent than the nucleic acid metabolites 2'd3'AMP and 3'AMP; b) explore whether, under what conditions, and to what extent the levels of 2'd3'AMP and 3"AMP may change in cells (e.g. in response to hormones and to changes in cell growth conditions, especially with conditions known to lower cellular cAMP content or to inhibit adenylyl cyclase); and c) isolate and characterize the enzyme catalyzing formation and degradation of 2'd3'AMP and 3'AMP. The biochemical approaches proposed are doable and will provide a unique handle on adenylyl cyclase. The highest priority will be to identify the sites(s) of inactivation by 2'5'dd3'FSBAdo. It is hoped that a new class of regulator of adenylyl cyclase will be defined, its source, fate, and binding site identified, and that a link will also have been established between regulation of adenylyl cyclase and regulation of nucleic acid metabolism.